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Antiphospholipid syndrome (APS) affects the brain by both hypercoagulation and immunological mechanisms. APS is characterized by several autoantibodies binding to a thrombolytic complex including beta-2-glycoprotein I (β2-GPI) and annexin A2 (ANXA2). Teriflunomide, an oral drug for the treatment of multiple sclerosis (MS), has a cytostatic effect on B cells and is therefore a potential antibody-targeting treatment for APS. In this study, we assessed the effect of teriflunomide in two APS mouse models by inducing autoantibody formation against β2-GPI and ANXA2 in female BALB/c mice. The ANXA2 model displayed a behavioral change suggesting an anti-anxiety effect in open field and forced swim tests, early in the course of the disease. This effect was normalized following teriflunomide treatment. Conversely, behavioral tests done later during the study demonstrated depression-like behavior in the ANXA2 model. No behavioral changes were seen in the β2-GPI model. Total brain IgG levels were significantly elevated in the ANXA2 model but not in the teriflunomide treated group. No such change was noted in the brains of the β2-GPI model. High levels of serum autoantibodies were induced in both models, and their levels were not lowered by teriflunomide treatment. Teriflunomide ameliorated behavioral changes in mice immunized with ANXA2 without a concomitant change in serum antibody levels. These findings are compatible with the effect of teriflunomide on neuroinflammation.Teriflunomide ameliorated behavioral and brain IgG levels in mice immunized with ANXA2 without a concomitant change in serum antibody levels. These findings are compatible with an effect of teriflunomide on the IgG permeability to the brain and neuroinflammation.

Introduction: Antiphospholipid syndrome (APS) is an autoimmune disorder manifested by thromboembolic events, recurrent spontaneous abortions and elevated titers of circulating antiphospholipid antibodies. In addition, the presence of antiphospholipid antibodies seems to confer a fivefold higher risk for stroke or transient ischemic attack. Although the major antigen of APS is β 2 glycoprotein I, it is now well established that antiphospholipid antibodies are heterogeneous and bind to various targets. Recently, antibodies to Annexin A2 (ANXA2) have been reported in APS. This is of special interest since data indicated ANXA2 as a key player in fibrinolysis. Therefore, in the present study we assessed whether anti-ANXA2 antibodies play a pathological role in thrombosis associated disease., Materials and Methods: Mice were induced to produce anti-ANXA2 antibodies by immunization with ANXA2 (iANXA2) and control mice were immunized with adjuvant only. A middle cerebral artery occlusion stroke model was applied to the mice. The outcome of stroke severity was assessed and compared between the two groups., Results: Our results indicate that antibodies to ANXA2 lead to a more severe stroke as demonstrated by a significant larger stroke infarct volume (iANXA2 133.9 ± 3.3 mm 3 and control 113.7 ± 7.4 mm 3 ; p = 0.017) and a more severe neurological outcome (iANXA2 2.2 ± 0.2, and control 1.5 ± 0.18; p = 0.03)., Conclusions: This study supports the hypothesis that auto-antibodies to ANXA2 are an independent risk factor for cerebral thrombosis. Consequently, we propose screening for anti-ANXA2 antibodies should be more widely used and patients that exhibit the manifestations of APS should be closely monitored by physicians.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies, which may trigger vascular thrombosis with consecutive infarcts. However, cognitive dysfunctions representing one of the most commonest neuropsychiatric symptoms are frequently present despite the absence of any ischemic brain lesions. Data on the structural and functional basis of the neuropsychiatric symptoms are sparse. To examine the effect of APS on hippocampal neurogenesis and on white matter, we induced experimental APS (eAPS) in adult female Balb/C mice by immunization with β2-glycoprotein 1. To investigate cell proliferation in the dentate gyrus granular cell layer (DG GCL), eAPS and control mice (n = 5, each) were injected with 5-bromo-2'-deoxyuridine (BrdU) once a day for 10 subsequent days. Sixteen weeks after immunization, eAPS resulted in a significant reduction of BrdU-positive cells in the DG GCL compared to control animals. However, double staining with doublecortin and NeuN revealed a largely preserved neurogenesis. Ultrastructural analysis of corpus callosum (CC) axons in eAPS (n = 6) and control mice (n = 7) revealed no significant changes in CC axon diameter or g-ratio. In conclusion, decreased cellular proliferation in the hippocampus of eAPS mice indicates a limited regenerative potential and may represent one neuropathological substrate of cognitive changes in APS while evidence for alterations of white matter integrity is lacking.

Aims: The antiphospholipid syndrome (APS) is an autoimmune disease characterized by high titres of auto-antibodies (aPL) leading to thrombosis and consequent infarcts. However, many affected patients develop neurological symptoms in the absence of stroke. Similarly, in a mouse model of this disease (eAPS), animals consistently develop behavioural abnormalities despite lack of ischemic brain injury. Therefore, the present study was designed to identify structural alterations of hippocampal neurones underlying the neurological symptoms in eAPS., Methods: Adult female Balb/C mice were subjected to either induction of eAPS by immunization with β2-Glycoprotein 1 or to a control group. After sixteen weeks animals underwent behavioural and cognitive testing using Staircase test (experiment 1 and 2) and Y-maze alternation test (experiment 1) and were tested for serum aPL levels (both experiments). Animals of experiment 1 (n = 7/group) were used for hippocampal neurone analysis using Golgi-Cox staining. Animals of experiment 2 (n = 7/group) were used to analyse molecular markers of total dendritic integrity (MAP2), presynaptic plasticity (synaptobrevin 2/VAMP2) and dendritic spines (synaptopodin) using immunohistochemistry., Results: eAPS mice developed increased aPL titres and presented with abnormal behaviour and impaired short term memory. Further, they revealed a reduction of dendritic complexity of hippocampal CA1 neurones as reflected by decreased dendritic length, arborization and spine density, respectively. Additional decrease of the spine-associated protein expression of Synaptopodin points to dendritic spines as major targets in the pathological process., Conclusion: Reduction of hippocampal dendritic complexity may represent the structural basis for the behavioural and cognitive abnormalities of eAPS mice., (© 2014 British Neuropathological Society.)

Mice immunized with β2-glycoprotein I (β2GPI) are an experimental model of the antiphospholipid syndrome (eAPS) displaying elevated titers of antiphospholipid antibodies (aPL). We presently studied whether the behavioral hyperactivity in eAPS mice is associated with in vivo binding and accumulation of IgG in the brain. At 6 weeks post immunization eAPS mice had significantly higher levels of aPL (1.32 ± 0.28 and 0.02 ± 0.01 AU, p < 0.001 by t-test) compared to adjuvant immunized controls, as measured by ELISA. Significant hyperactivity in a staircase test in the eAPS mice compared to controls was found in stair-climbing (18.4 ± 0.9 and 12.0 ± 1.7, respectively) and rearing measures (23.5 ± 2.1 and 12.5 ± 1.9, p < 0.01 by t-test). Immunofluorescence staining in eAPS mice revealed significant in vivo accumulation of IgG in cortical and hippocampal neurons which was not seen in controls. Staining for IgG was markedly intense in inhibitory interneurons co-stained for GAD67 in the hippocampus of eAPS mice. The integrity of the blood brain barrier (BBB) evaluated by injection of Evans blue (EB) was impaired in eAPS and adjuvant immunized mice compared to naïve mice. Electrophysiological recordings in hippocampal brain slices showed altered response to paired pulse stimulation as well as dysregulation of carbachol-induced γ- oscillations in eAPS mice compared to control. Penetration into the brain and direct interaction of aPL with inhibitory interneurons in the hippocampus may explain the hyperactive behavior of the eAPS mice. A direct role of aPL in causing CNS dysfunction points to these antibodies as an important therapeutic target in APS., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Experimental antiphospholipid syndrome (eAPS) in Balb/c mice causes neuropsychiatric abnormalities including hyperactivity, increased explorative behavior and cognitive deficits. Recently, we have demonstrated that these behavioral changes were linked to an upregulation of serotonergic 5-HT1A receptor binding densities in cortical and hippocampal regions while excitatory and inhibitory neurotransmitter receptors remain largely unchanged. To examine whether the observed behavioral features depend on a critical antibody concentration, mice with only moderately enhanced antiphospholipid antibodies (aPL), about 50-80% of high levels, were analyzed and compared to controls. The staircase test was used to test animals for hyperactivity and explorative behavior. The brains were analyzed for tissue integrity and inflammation. Ligand binding densities of NMDA, AMPA, GABAA, 5-HT1A, M1 and M2 muscarinic acetylcholine receptors, respectively, were analyzed by in vitro receptor autoradiography and compared to brains of mice from our previous study with high levels of aPL. Mice with only moderately enhanced aPL did not develop significant behavioral changes. Brain parenchyma remained intact and neither inflammation nor glial activation was detectable. However, there was a significant decrease of NMDA receptor binding densities in the motor cortex as well as an increase in M1 binding densities in cortical and hippocampal regions, whereas the other receptors analyzed were not altered. Lack of neuropsychiatric symptoms may be due to modulations of receptors resulting in normal behavior. In conclusion, our results support the hypothesis that high levels of aPL are required for the manifestation of neuropsychiatric involvement while at lower antibody levels compensatory mechanisms may preserve normal behavior., (Copyright © 2013. Published by Elsevier GmbH.)

Background: We investigated interactions between genetically and autoimmune-mediated coagulopathies by inducing experimental antiphospholipid syndrome (eAPS) in mice carrying the factor V Leiden (FVL) mutation., Methods: eAPS was induced in heterozygous and homozygous FVL transgenic mice (C57BL/6 background) by immunization with β(2)-glycoprotein I (β(2)-GPI). Autoantibody levels were measured at 1 and 5 months post-immunization. Mice were tested at 4 months post-immunization for behavior and cognitive function in the staircase, elevated plus-maze, and swim T-maze tests. Brains were removed and analyzed by immunohistochemistry for inflammatory markers and neurodegenerative processes., Results: A single immunization with β(2)-GPI induced significantly higher and longer-lasting immune responses, and this was dependent on the number of FVL alleles. At 1 and 5 months post-immunization, levels of antibodies rose from 1.17 ± 0.07 to 1.62 ± 0.17 (optical density units; ODU) in homozygous FVL mice, compared with stable levels of 0.59 ± 0.17 and 0.48 ± 0.16 ODU in heterozygous FVL mice and a drop from 1.62 ± 0.21 to 0.61 ± 0.13 ODU in wild-type mice. Behavioral and cognitive clinical features of eAPS were also correlated with FVL allele load, as assessed by the elevated plus-maze (altered anxiety), staircase (hyperactivity and higher exploration), and swim T-maze (impaired learning) tests. Histological studies identified significant neurodegenerative changes in both grey and white matter in the eAPS-FVL brains. In spite of the potential interaction of two prothrombotic disease states, there were no ischemic lesions seen in this group., Conclusions: The results indicate that genetically mediated coagulopathies increase the risk of developing coagulation-targeted autoimmune responses, and suggest the importance of antibody-mediated neurodegenerative processes in the brain in APS.

The antiphospholipid syndrome (APS) is an autoimmune disease where the presence of high titers of circulating autoantibodies causes thrombosis with consecutive infarcts. In experimental APS (eAPS), a mouse model of APS, behavioral abnormalities develop in the absence of vessel occlusion or infarcts. Using brain hemispheres of control and eAPS mice with documented neurological and cognitive deficits, we checked for lymphocytic infiltration, activation of glia and macrophages, as well as alterations of ligand binding densities of various neurotransmitter receptors to unravel the molecular basis of this abnormal behavior. Lymphocytic infiltrates were immunohistochemically characterized using antibodies against CD3, CD4, CD8 and forkhead box P3 (Foxp3), respectively. GFAP, Iba1 and CD68-immunohistochemistry was performed, to check for activation of astrocytes, microglia and macrophages. Ligand binding densities of NMDA, AMPA, GABAA and 5-HT1A receptors were analyzed by in vitro receptor autoradiography. No significant inflammatory reaction occurred in eAPS mice. There was neither activation of astrocytes or microglia nor accumulation of macrophages. Binding values of excitatory and inhibitory neurotransmitter receptors were largely unchanged. However, ligand binding densities of the modulatory serotonergic 5-HT1A receptors in the hippocampus and in the primary somatosensory cortex of eAPS mice were significantly upregulated which is suggested to induce the behavioral abnormalities observed., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

The antiphospholipid syndrome (APS) is characterized by antiphospholipid antibodies (aPL) and vascular brain disease which is often associated with dementia. We examined the neurodegenerative pathological processes underlying APS by inducing APS in a transgenic animal model of Alzheimer's disease. Female C57B6/SJL mice carrying the APP(695)SWE mutation (Tg2576) and wild-type (wt) controls were immunized with β₂-glycoprotein-I (APS mice) or adjuvant alone (controls) at 4 months of age. At the age of 8 months the APP-APS mice developed high levels of aPL associated with motor hypoactivity in a staircase test (p<0.03 by t-test) and impaired performance in the cognitive T-maze (p<0.02 for main effect of treatment by repeated measures ANOVA) relative to APP-CFA mice and controls. wt-APS and wt-control did not differ significantly in their behavior or cognition. Histological studies revealed mature plaques only in the APP-APS group which also had higher amyloid load and number of activated microglia compared to all other groups. The results indicate a significant interaction between APP genotype and the induction of APS on a female background. The mechanisms involved may also be important in human APS-AD co-morbidity., (Copyright © 2009 Elsevier Inc. All rights reserved.)

Circulating antiphospholipid antibodies (aPL) are associated with central nervous system dysfunction in antiphospholipid syndrome (APS) patients and in a mouse model of APS. We propose a logical pathway of how experimental APS (eAPS) causes brain dysfunction: binding of the antibodies to the brain endothelium evoking microthrombosis, endothelial dysfunction, and IgG leakage through the blood-brain barrier (BBB), then secondary inflammatory cell spread around blood vessels and production of cytokines by these inflammatory cells leading to further disruption of the BBB. The diffuse brain endothelial dysfunction would result in extravasation of serum proteins including APS IgG and activated thrombin, which may induce the behavioral changes observed in the APS mice. We have collected data from the mouse eAPS model which supports this hypothesis. Elucidating the mechanism of the pathogenicity of aPL in vitro and in vivo will serve as a much needed basis for developing new therapeutic modalities in this important disorder.

The antiphospholipid syndrome (APS) is characterized by recurrent arterial and/or venous thromboses, pregnancy loss and the presence of antiphospholipid antibodies (aPL). One of the discussed mechanisms of this thrombotic activity in APS patients is attributed to TNFalpha secretion in monocytes after aPL stimulation. To investigate this mechanism in detail, we employed a monoclonal aPL and IgG fractions of APS patients for stimulation of human peripheral monocytes. Stimulation with this monoclonal aPL resulted in an increased expression and secretion of TNFalpha, caused by specific upregulation of TLR8 mRNA and protein expression levels. To confirm the specificity of this finding we could demonstrate that the TNFalpha enhancement could be neutralized by a TLR8-specific inhibitory DNA-oligonucleotide and could be further increased by adding the specific ligands for TLR8. Using APS patients IgG fractions for stimulation of peripheral monocytes revealed a similar TLR8 mRNA elevation and increase in TNFalpha-production. Furthermore the TLR8 expression level in PBMC's of APS patients was as well significantly elevated. It could be demonstrated that the TNFalpha release in monocytes resulting from aPL stimulation was exclusively induced by TLR8 engagement. This could be confirmed in PBMC's of APS patients, hinting that endogenous stimulation of TLR8 in APS patients and consecutive elevation of TNFalpha promotes a proinflammatory environment., (2009 Elsevier GmbH. All rights reserved.)

Antiphospholipid syndrome (APS) is a multisystem disorder characterized by arterial and venous thrombosis, pregnancy morbidity, and neuropsychiatric manifestations. Antiphospholipid IgG injected intracerebroventricularly (i.c.v.) cause behavioral hyperactivity in mice. In the present study we investigated the effects of APS whole-serum i.c.v. administration in female Balb/C mice. Control mice were injected with serum derived from healthy subjects or saline solution. Behavior was assessed by the staircase apparatus which combines locomotor (stair-climbing) exploratory activities and rearing as a measure of anxiety. Mice injected with serum from APS patients or serum from normal subjects showed a trend to an increase in the number of stairs climbed in the APS group. The results suggest a differential effect of specific IgG and other serum components in the CNS manifestations of APS.

Experimental antiphospholipid syndrome (eAPS) induced by immunization with beta(2)-glycoprotein I (beta(2)-GPI) causes behavioral hyperactivity. We assessed the role of thrombotic and inflammatory perivascular factors and standard APS therapies for CNS manifestations. Groups of mice (n=10 per group) were immunized once with beta(2)-GPI (eAPS) or adjuvant (controls) and treated daily from 1 month after immunization with either sham injections, aspirin (1.2 mg/kg) or enoxaparin (1 mg/kg) for 3 months. Serum antiphospholipid antibodies (aPL) and brain levels of tissue necrosis factor-alpha (TNF-alpha) and prostaglandin E (PGE) were then measured by ELISA and thrombin inhibitors by immunoblot. Behavioral hyperactivity was assessed by the staircase test. The eAPS mice had higher levels of aPL than adjuvant immunized controls. Inflammatory markers were found to be twofold higher and intrinsic brain thrombin inhibitors 50% lower in eAPS brains compared to controls. aPL titers were unaffected by treatment. Both aspirin and enoxaparin normalized brain concentrations of PGE and TNF-alpha and elevated thrombin inhibitors, the latter effect being more pronounced for enoxaparin. The increased activity and rearing exploratory behavior in eAPS (138.6+/-13.6 and 141.9+/-13.9% of controls, respectively) were attenuated significantly more by treatment with enoxaparin (91.5+/-12.3 and 95.0+/-9.8%) than by aspirin (167.0+/-18.4 and 114.7+/-13.1%, p<0.01, ANOVA). Together, these results demonstrate that eAPS is associated with significant brain inflammation and thrombosis that are well treated with both standard therapies for human APS. Enoxaparin attenuates behavior better than aspirin possibly by reducing thrombosis or stabilization of the blood brain barrier, suggesting an advantage for similar drugs in CNS APS.

Objective: We sought (1) to identify and (2) to define the association of all reported antibodies (Abs) with neuropsychiatric lupus (NPSLE), (3) to search for possible mechanisms that are involved in NPSLE, and (4) to determine whether we can recognize a panel of Abs associated with specific neuropsychiatric (NP) manifestations., Methods: A MEDLINE search (1975 to 2005) was performed utilizing the following terms: neuropsychiatric lupus, antiphospholipid syndrome, or central nervous system systemic lupus erythematosus matched with the term antibodies., Results: Twenty Abs (11 brain-specific and 9 systemic) were described in NPSLE patients. These include Abs that target brain-specific antigens (neuronal, ganglioside, synaptosomes, glia, methyl-d-aspartate receptors, lymphocytotoxic) and systemic antigens (nuclear, cytoplasmic, phospholipid, endothelial cells). Cognitive impairment, psychosis, and depression were associated with many Abs. Elevated titers of anticardiolipin Abs (aCL) were reported most often and found in patients with cognitive impairment, psychosis, depression, seizures, chorea, and migraine. No specificity was encountered among brain-specific or systemic Abs for any single NP manifestation. No studies evaluated a specific NP manifestation with the full panel of 20 Abs. A panel of brain-specific and systemic Abs may be helpful in establishing the diagnosis of NPSLE. Postulated mechanisms in experimental models included vascular occlusion and injury by pathogenic Abs in a disrupted blood brain barrier., Conclusions: NPSLE is associated with brain-specific and systemic Abs. Cognitive impairment, psychosis, and depression were associated with many Abs, including aCL Abs. Possible mechanisms include vascular occlusion and injury by pathogenic Abs in a disrupted blood brain barrier.

The antiphospholipid syndrome (APS) includes systemic and central nervous system (CNS) pathology associated with antibodies to a complex of phospholipids and beta(2)-glycoprotein I (beta(2)-GPI). We have recently reported the induction of APS associated with behavioral and cognitive deficits in BALB/c female mice that developed 4-5 months after immunization with beta(2)-GPI. In the present study, we examined the influence of genetic factors on the ability to induce experimental APS with CNS involvement by testing several mouse strains immunized with beta(2)-GPI. Female mice from five strains were immunized once with beta(2)-GPI in complete Freund's adjuvant (CFA) or with CFA alone (controls). Autoantibody levels were examined at 1 and 5 months after immunization. Neurological assessment in a staircase test was performed 4-5 months following the immunization. Induction of APS resulted in elevated levels of antibodies against negatively charged phospholipids and beta(2)-GPI in all five mouse strains. Autoantibody levels were significantly higher in Balb/c, ICR, and C57BL/6 mouse strains compared to AKR and C3H. aPL levels dropped significantly more in the C57BL/6 compared to Balb/c mice over a period of 4 months. Hyperactivity reflected by higher number of stairs climbed in 3 min, was induced by APS in the Balb/c and ICR, mouse strains. Exploratory behavior reflected by more frequent rears, was seen in the APS-Balb/c and AKR mice. Hypoactivity and less exploration were seen in the APS-C57BL/6 and C3H mice. The study supports a link between high levels of aPL and behavioral changes in a mouse APS model. Qualitative differences in behavioral patterns may be due to nervous system as well as immune genetic factors. The minimal effect of APS in C57BL/6 mice may provide a suitable background for the study of transgenes in these mice.

We report a 36-year-old woman with the occurrence of painful focal seizures of her left hand and the left leg. She also had focal motor seizures at the left corner of her mouth. The duration and frequency of the episodes increased over four days from a few seconds once a day to frequent intervals lasting more than four hours at a time. The symptoms appeared one day after start of the treatment with fluvastatin (40 mg) administered in order to diminish the endothelial activation induced by antiphospholipid antibodies (aPL). The patient suffered from severe manifestations of the antiphospholipid syndrome (APS) including Catastrophic Antiphospholipid Syndrome (CAPS, Asherson's syndrome). In this case a single 40 mg dose of oral fluvastatin was linked to seizures. After discontinuation of this treatment, the seizures immediately disappeared and the patient fully recovered without evidence of permanent neurological damage. This data links statins to seizures in patients with compromised blood brain barrier such as APS.

Objective: To assess the frequency of epilepsy in primary and secondary antiphospholipid syndrome (APS); to analyze the clinical and laboratory features characterizing those with epilepsy in a cohort of 538 patients with APS; and to find associated features that would suggest risk factors for epilepsy in APS., Methods: We analyzed the clinical features of patients with APS who had epilepsy and compared them to the clinical features of non-epileptic APS patients., Results: Of 538 APS patients, 46 (8.6%) had epilepsy. Epilepsy was more prevalent among APS secondary to systemic lupus erythematosus (SLE) compared to primary APS (13.7% vs 6%; p < 0.05). The patients with epilepsy had a higher prevalence of central nervous system (CNS) manifestations including focal ischemic events (strokes or transient ischemic events, 54.3% vs 24.6%; p < 0.0001) and amaurosis fugax (15.2% vs 4.9%; p < 0.05). APS patients with epilepsy had a higher frequency of valvular pathology (30.4% vs 14.6%; p < 0.01), thrombocytopenia (43.5% vs 25%; p < 0.05), and livedo reticularis (26.1% vs 11.5%; p < 0.01). The multivariate logistic regression analysis found CNS thromboembolic events as the most significant factor associated with epilepsy, with an odds ratio (OR) of 4.05 (95% confidence interval, CI: 2.05-8), followed by SLE (OR 1.4, 95% CI 1.2-4.7), and valvular vegetations (OR 2.87, 95% CI 1-8.27)., Conclusion: Epilepsy is common in APS and most of the risk seems to be linked to vascular disease as manifested by extensive CNS involvement, valvulopathy, and livedo reticularis and to the presence of SLE. These factors, however, explain only part of the increased occurrence of epilepsy in APS and other causes such as direct immune interaction in the brain should be investigated.

The aim of this work was to study the pathological processes underlying neurological dysfunctions displayed by BALB/C mice induced with experimental antiphospholipid syndrome (APS), as we have previously reported. Experimental APS was induced in female BALB/C mice by immunization with a pathogenic monoclonal anticardiolipin (aCL) antibody, H-3 (n = 10), or an irrelevant immunoglobulin in controls (n = 10). Mice immunized with H-3 developed clinical and neurological manifestations of APS, including: embryo resorption, thrombocytopenia neurological defects and behavioral disturbances. In mouse sera, the titer of various autoantibodies were elevated, including: anti-phospholipids (aPLs), anti-beta2 glycoprotein-I (beta2GPI), anti-endothelial cell antibodies (AECA) and low titer of anti-dsDNA antibodies. Five months after APS induction, mice were sacrificed and brain tissue specimens were processed for hematoxylin and eosin (H&E), immunofluorescence staining and transmission electron microscopy (TEM). H&E staining of cortical tissue derived from all APS mice revealed mild inflammation, localized mainly in the meninges. Prominent IgG deposits in the large vessel walls and perivascular IgG leakage were observed by immunofluorescence. No large thrombi were observed in large vessels. However, EM evaluation of cerebral tissue revealed pathological changes in the microvessels. Thrombotic occlusion of capillaries in combination with mild inflammation was the main finding and may underlie the neurological defects displayed by mice with APS.

The interface between multiple sclerosis (MS) and the antiphospholipid syndrome (APS) is of great clinical relevance and may also shed some light on their pathogenesis. Although formal diagnostic criteria of these two immune syndromes are ostensibly mutually exclusive, many patients with subcortical white matter brain lesions share features of both MS and APS. The main diagnostic feature of APS, the antiphospholipid antibodies (aPLAb), is relatively common in multiple sclerosis and increased fibrin deposition is a feature of both APS lesions and MS plaques. A closer examination of the clinical and laboratory data reveals, however, fundamental differences between the syndromes: MS is a diffuse disease of white matter compared to a much more patchy disease in APS as demonstrated by recent studies in imaging and electrophysiology. Furthermore, a closer examination of the specific aPLAb profile reveals significant differences: we have found that although aPLAb were common in MS patients (32%), these antibodies did not depend on the presence of serum factors for binding to phospholipids and none of the MS patients had antibodies to beta2-glycoprotein-I (beta2-GPI), the major autoantigen in APS. Animal models of the two syndromes are induced by very different antigens and may serve to assess overlapping pathogenic mechanisms. The most common treatment used in MS are beta-interferons which theoretically may exacerbate APS, while anticoagulation, the mainstay of APS treatment, is of unknown value in MS. The relationship of MS to APS remains to be clarified by focused large collaborative studies.

Background: Stimulation and proliferation of lymphocytes require activation of Ras. S-farnesylthiosalicylic acid (FTS) is a synthetic substance that detaches Ras from the inner cell membrane and induces its rapid degradation. Antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies detected in patients with antiphospholipid syndrome (APS), which is associated with thrombosis, pregnancy losses, and thrombocytopenia., Objective: To examine the effect of FTS treatment on aPL levels in a genetic autoimmune model (the MRL/lpr mice) and in an induced model of APS., Methods: Female Balb/C mice immunized once with beta2-glycoprotein I (beta2-GPI) in complete Freund's adjuvant (CFA) and female MRL/lpr mice were treated intraperitoneally with either FTS (5 mg/Kg/day) or saline 3-5 times a week. aPL and anti-beta2-GPI antibodies were measured by ELISA., Results: FTS treatment 3 times a week resulted in significant decreases of aPL and anti-beta2-GPI antibodies in both animal models. In contrast, more frequent treatment (5 times a week) had no significant effect on autantibody levels in both animal models. We further compared 2 protocols in the induced APS model, one for alternate day treatment and the other for daily treatment on the first 3 days each week, and found a decrease in autoantibody levels only in the alternate day protocol., Conclusions: Inhibition of Ras activation by FTS is effective in decreasing autoantibody levels in models of APS. The differential modulation of immune function by alternate day compared to daily treatment may provide better understanding of the role of Ras activation in this system.

The increasing prevalence with age of antiphospholipid antibodies (aPL), of dementia and of stroke complicates the study of a causal relationship between antiphospholipid syndrome (APS) and dementia. Prolonged aPTT due to circulating anticoagulants (CAC) may serve as a more specific laboratory marker of APS. In a hospital-based study, we examined all patients with CAC and included 23 who fulfilled standard criteria for primary APS. These patients were assessed for dementia, vascular brain disease, autoimmune disease activity and dementia risk factors. Among CAC-positive APS patients, 13 of the 23 (56%) were demented and these were significantly older (mean age+/-S.E., 68+/-3 years) than the nondemented APS group (n=10, 51+/-4 years; p<0.01, Student's t-test). The demented patients had significantly more pathology on computerized brain tomography (CT) and electroencephalography (EEG) studies but six of them had no clinical or CT evidence of vascular brain disease. Erythrocyte sedimentation rate was significantly lower in the dementia group, in which there was also a significant negative correlation between levels of aPL and age. CAC-positive APS patients seem to be at risk for developing dementia with age, suggesting a pathogenic role for prolonged exposure to elevated aPL.

Although many neurological deficits have been described in the antiphospholipid syndrome (APS), only stroke is well established and accepted as a diagnostic criterion in the disease. We presently review clinical data obtained from large series of cases regarding stroke, dementia, epilepsy, chorea, migraine, white-matter disease, and behavioral changes in APS, or linked-to-laboratory criteria such as antiphospholipid antibodies (aPL). The contribution of animal models to our understanding of these manifestations of APS is stressed, especially regarding the cognitive and behavioral aspects for which we have established model systems in the mouse. These models utilize immunization of mice with beta(2)-glycoprotein1, a central autoantigen in APS, which induces persistent high levels of aPLs. These mice develop hyperactive behavior after a period of four months, as well as deficits in learning and memory, and are potentially valuable as a system in which to study the pathogenesis and treatment of cognitive and behavioral aspects of APS. We have developed another model, in which IgGs from APS patients induce depolarization of brain synaptoneurosomes, and which may serve as a model for the pathogenesis of epilepsy in APS. Hormonal changes are another potential CNS manifestation of APS and this may be potentially linked to the systemic and central effects of cytokines such as interleukin-3. Better understanding of the link between APS and neurological or neuroendocrine manifestations other than stroke will reveal whether they can be used as clinical criteria for the diagnosis of APS and, it is hoped, lead to better treatment.

Thrombin and other proteases involved in coagulation also have the potential to stimulate inflammation in the brain to a large extent through the protease-activated receptors (PARs). Such exposure of the brain to increased levels of coagulation factors is more likely to occur during vasculitis and activation of intrinsic coagulation in the brain and may cause inflammatory diseases such as multiple sclerosis. There is growing evidence from animal models and human brain samples that links upregulation of coagulation factors to inflammatory central nervous system (CNS) disease. Evidence includes measures of thrombin-like activity, levels of its receptors, PARs, and inhibitors of the coagulation pathway. The major receptor for thrombin, PAR-1, has now been definitively located to the synapse and node of Ranvier and its excessive activation leads to blocks in long-term potentiation and in nerve conduction. There is growing evidence that modulating coagulation in models of CNS inflammatory disease has beneficial clinical effects. These findings suggest that coagulation-like pathways play a significant role in the pathogenesis of inflammatory diseases in the CNS and present a viable target for therapeutic strategies. [ABSTRACT FROM AUTHOR]

Antiphospholipid syndrome (APS) is characterized by thromboses and neuropsychiatric manifestations possibly linked to brain inflammation. In order to examine the levels of proinflammatory and anti-inflammatory cytokines in experimental APS (eAPS) mice brains, we measured the levels of TNF-α, IFN-γ, and IL-10 in brain homogenates (cytosolic fractions) and in brain slices (secreted level) at 6, 15, and 24 weeks after immunization. We induced eAPS by immunization of Balb/c mice with β2-glycoprotein I (β2GPI), the major autoantigen in the disease and controls with adjuvant alone. We found increased levels of secreted TNF-α in eAPS mice for the entire experiment period. Cytosolic and secreted IL-10 and IFN-γ levels in eAPS mice were lower at 6 and 15 weeks and higher at 24 weeks after immunization. The results suggest that brain disease in APS is associated with significant and complex changes in proinflammatory and anti-inflammatory cytokines. [ABSTRACT FROM AUTHOR]

β2-glycoprotein-I (β2-GPI, also known as apolipoprotein H) is a major autoantigen in the antiphospholipid syndrome (APS), a disease commonly affecting the central nervous system. We examined whether β2-GPI and similar proteins exist in rat and human brains. No expression was found on Northern blot analysis of human brain. Utilizing a standard procedure for the isolation of serum β2-GPI we purified a 100 kD human brain protein, which was found by peptide sequencing to have full homology with the serum protein, histidine-rich glycoprotein (HRGP). Expression of HRGP in rat and human brain was established by RT-PCR studies and a partial sequence of rat brain HRGP was obtained showing 68% homology with the human protein. IgG from most APS patients bound to HRGP, which shares distinct biochemical properties with β2-GPI, is present in the brain and may be an important autoantigen. [ABSTRACT FROM AUTHOR]

Abstract: The antiphospholipid syndrome (APS) is an autoimmune disease where the presence of high titers of circulating autoantibodies causes thrombosis with consecutive infarcts. In experimental APS (eAPS), a mouse model of APS, behavioral abnormalities develop in the absence of vessel occlusion or infarcts. Using brain hemispheres of control and eAPS mice with documented neurological and cognitive deficits, we checked for lymphocytic infiltration, activation of glia and macrophages, as well as alterations of ligand binding densities of various neurotransmitter receptors to unravel the molecular basis of this abnormal behavior. Lymphocytic infiltrates were immunohistochemically characterized using antibodies against CD3, CD4, CD8 and forkhead box P3 (Foxp3), respectively. GFAP, Iba1 and CD68-immunohistochemistry was performed, to check for activation of astrocytes, microglia and macrophages. Ligand binding densities of NMDA, AMPA, GABAA and 5-HT1A receptors were analyzed by in vitro receptor autoradiography. No significant inflammatory reaction occurred in eAPS mice. There was neither activation of astrocytes or microglia nor accumulation of macrophages. Binding values of excitatory and inhibitory neurotransmitter receptors were largely unchanged. However, ligand binding densities of the modulatory serotonergic 5-HT1A receptors in the hippocampus and in the primary somatosensory cortex of eAPS mice were significantly upregulated which is suggested to induce the behavioral abnormalities observed. [Copyright &y& Elsevier]

Abstract: Background: The antiphospholipid syndrome (APS) commonly affects the central nervous system through mechanisms that may include small vessel pathology and activation of thrombin. Antiphospholipid antibodies (aPL) activate endothelial cells but the specific activation of brain vascular endothelial cells (BVEC) and the receptors and signaling pathways involved have not been fully characterized. Objective: To examine whether aPL, the inflammatory stimulant lipopolysacharide (LPS) and thrombin activate BVECs through a Ras-dependent pathway. Methods: Rat BVEC (G8) were grown to confluence on 24-well plates. IgG was purified from 8 APS patients on a protein G column. Phosphorylation of ERK in the BVEC was measured by immunoblot utilizing a specific antibody. Results: Significant phosphorylation of ERK was measured following exposure of the cells to LPS and thrombin and this was blocked by the Ras inhibitor farnesylthiosalicylate (FTS). aPL IgG (1:100 relative to serum) from 7/8 patients also induced phosphorylation of ERK. Conclusions: Activation of the Ras-ERK pathway is an effect of both APS IgG and thrombin. This pathway is potentially amenable to drugs such as FTS and may serve as a therapeutic target in APS. [Copyright &y& Elsevier]